The ubiquitin (Ub)-proteasome system is responsible for the majority of intracellular protein degradation in eukaryotes. Both specific regulatory proteins and a broad range of misfolded or otherwise abnormal proteins must be recognized. Many diseases, including neurodegenerative disorders, diabetes, cystic fibrosis, and many types of cancer, are associated with abnormalities in the Ub system. Therefore, an understanding of how proteolytic substrates are recognized and covalently modified by Ub is expected to have a substantial impact on our ability to diagnose and treat many serious human medical conditions. In this renewal, a series of basic studies on Ub-dependent proteolysis is proposed. The focus is on the prototype natural substrate Mata2, a transcriptional regulator in the yeast S. cerevisiae. Mata2 is ubiquitinated in vivo by two distinct mechanisms. One depends on the Ubc6 and Ubc7 Ub-conjugating enzymes (E2s) and the Doa10 Ub ligase (E3), and the other on the Ubc4 E2. An Ubc6-Ubc7-Doa10 ubiquitination complex is believed to assemble on the endoplasmic reticulum (ER) or nuclear envelope (NE) and to recognize Mata2 through a Mata2 domain called the Deg1 degron. Neither the degron nor the E3 for the Ubc4 pathway is known; experiments are proposed in this application to identify both. In the remainder of the application, emphasis is placed on the transmembrane Doa10 Ub ligase, which targets both the soluble, nuclear Mata2 protein and ER membrane proteins. The overall goal is to define the key biochemical and cell biological determinants of specificity for this conserved ER/NE membrane-embedded Ub-ligation system. Evidence for a major role for Doa10 and its orthologs in ER physiology is beginning to emerge, so these experiments are expected to shed light on ER-associated and nuclear protein degradation in organisms ranging from yeast to human.